Composition comprising at least to compounds which induces indolamine 2,3 - dioxygenase (ido), for the treatment of an autoimmune disorder or suffering from immune rejection of organs

ABSTRACT

A composition and method for using a composition, the composition having at least two compounds, each of which induces indolamine 2,3-dioxygenase, for the treatment of an autoimmune disorder or disease or immune rejection of transplants or gene therapeutically modified cells, wherein the inducers have different mechanism of action and wherein the composition gives rise to a synergistic effect on the IDO levels.

FIELD OF INVENTION

The invention relates to the use of a composition comprising at leasttwo compounds, each of which induces indolamine 2,3-dioxygenase, for thetreatment of an autoimmune disorder or disease or immune rejection oftransplants or gene therapeutically modified cells, wherein saidinducers have different mechanism of action and wherein the compositiongives rise to a synergistic effect on the IDO levels.

BACKGROUND OF INVENTION

Indoleamine dioxygenase (IDO) degrades the indole moiety of tryptophanand initiates the production of neuroactive and immunoregulatorymetabolites, collectively known as kynurenines. The functionalexpression of IDO by dendritic cells has emerged in recent years as amajor mechanism of peripheral tolerance. IDO contributes to maternaltolerance in pregnancy, control of allograft rejection, and protectionagainst autoimmunity, inflammatory pathology and allergy. IDO expressionalso serves a physiological mechanism by which malignancies induceimmune tolerance (Uyttenhove et al. 2004; Mellor et al. 2004; Munn etal. 2004). The wide spectrum of physiopathological conditions in whichIDO appears at work suggests that this suppressive system is frequentlyinvolved in physiological down regulation of T cell responses andresulting inflammatory responses. There are a number of known substancesthat induces IDO, wherein said compounds have different mechanisms ofaction. Examples of classes of such IDO inducers, having differentmechanisms of action, are among others cytidine analogues, histonedeacetylase inhibitors, vitamin D3 analogues, interferons, toll likereceptor ligands, gonadotropine receptor signalling hormones,prostaglandine E2 analogues, IDO stabilisers, soluble CTLA4 conjugates,and glycocorticoids.

However, many of these substances increase the amount of IDO to levelswhich are too low to be suitable in pharmaceutical composition, and willthus require, to induce effective IDO levels, high doses that are notsuitable for reasons of toxicology, compliance or costs. Therefore thereis a need to develop new pharmaceutical compositions that, at suitabledose levels, could increase IDO to levels that are sufficient andtherapeutically useful in the treatment of different autoimmunedisorders and in the prevention of transplant rejections.

SUMMARY OF THE INVENTION

The invention relates to the finding that compounds, that, when usedalone, induces IDO to levels that are not sufficient in relation to thetreatment regimes, could be used in mixtures or combinations ofcompounds that induce IDO, and that by mixing different IDO inducershaving different mechanism of action, the increase in IDO was more thanthe sum of what each IDO inducer would have achieved alone, and in somecases up to 100 times larger than that additive effect. By making such acombination it will for the first time be possible to produce apharmaceutical composition which could be used for the treatment of amammal in need thereof for a number of diseases and disorders in whichIDO induction is therapeutically useful.

In a first aspect the invention relates to a composition comprising atleast two compounds, which induces IDO, for the treatment of anautoimmune disorder or disease or immune rejection of transplants orgene therapeutically modified cells, wherein said inducers havedifferent mechanisms of action and give rise to a synergistic effect onthe IDO level.

In a second aspect, the invention relates to a method of treating amammal having an autoimmune disorder or disease or having an immunerejection of transplants or gene therapeutically modified cells, whereinthe treatment induces IDO, comprising administering to a patient atherapeutically effective amount of the composition as defined above.

In a third aspect of the invention, dendritic cells or other antigenpresenting cells, for example from peripheral blood or bone marrow ofthe patient or of another person, are cultered ex vivo in a suitablemedium. To these cells are added the invented composition with the aimto induce IDO production and induce differentiation to cells that havean elevated IDO production. Simultaneously or soon thereafter, one ormore antigens, that are associated with the condition being treated(e.g., an autoantigen responsible for an autoimmune disease) will beadministered to the cells, whereafter the cells are transferred to thepatient. This ex vivo treatment, or adoptive cell transfer as it mayalso be called in the scientific literature, will lead to a migration ofthe transferred cells to organs or tissues where they can activateT-cells to become suppressive T-cells or to become antigen-specificregulatory T-cells. The transferred cells can also migrate to sites ofinflammation where they can locally perpetuate existing regulatoryT-cells.

In a fourth aspect the invention relates to a method of inducing IDO ina cell culture comprising the steps of; providing isolated cells in asuitable medium, adding the composition as defined above, incubatingsaid isolated cells with the composition and obtaining a cell culture inwhich IDO is induced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Synergistic effect on IDO expression by zebularine andinterferon gamma The scale is logaritmic and the relative values aregiven for each bar.

FIG. 2: Synergistic effect on IDO expression by interferon gamma andvalproic acid. 2A: Valproic acid at 1 mM. 2B: Valproic acid at 0.5 mM.

FIG. 3: Synergistic effect on IDO expression by zebularine, interferongamma and valproic acid. The result clearly demonstrates a synergisticeffect of all the three substances on the IDO1 expression by THP-1cells.

FIG. 4: Synergistic effect on IDO expression by hCG and zebularine andby hCG and interferon gamma 4A: Cells of the human monocytic cell lineTHP-1 were non-exposed (medium control), exposed to zebularine alone(100 uM), hCG alone (0.1 units/ml), or a combination. The results ofTHP-1 cells exposed to the combination demonstrate a synergistic effect.4B: Results obtained when cells were non-exposed (medium control),exposed to hCG alone (0.01 units/ml), inteferon gamma alone (200 IU/ml),or a combination. The THP-1 cells exposed to the combinationdemonstrated a synergistic effect.

FIG. 5: Kinetics of IDO1 expression after exposure to interferon gammafor 24 hours from start and continuous exposure to zebularine. The aimof the study was to investigate the kinetics of interferon incombination with zebularine. The IDO1 induction by interferon gammaalone was strong after 24 hours but the effect was not sustained anddropped rapidly. In contrast, when combined with zebularine maintainedfor the whole period, the effect was sustained at days 2 and 3, althoughat a reduced level.

FIG. 6: Kinetics of IDO1 expression after interferon gamma exposure for24 hours after different pre-exposure times to zebularine. Kinetics ofsynergistic effect on IDO expression by zebularine and interferon gamma.In panel A we demonstrate a low effect of zebularine after four days.Interferon gamma alone given after 3 days, 24 hours before harvest givesa strong band and when interferon gamma is added after 3 days, 24 hoursbefore harvest of THP-1 cells exposed to zebularine, it gives a strongereffect, demonstrating a synergistic effect also when zebularine preceedsthe interferon with 3 days. In panel B, the THP-1 cells were givenzebularine alone during 5 days and a weak band was detected. Interferongamma alone given after 3 days and washed away 24 hours later, showed aweak band when tested 24 hours after removal of interferon. This is inagreement with results presented in FIG. 5. The combination ofzebularine from start and interferon gamma given for 24 hours afterthree days, resulted in a sustained high expression level. In panel Cthe THP-1 cells were exposed to zebularine for 6 days and interferongamma was given during 24 hours after four days and the cells wereharvested after a total of 6 days. Again almost no IDO1 induction byinterferon gamma alone was detected, but with the combination a strongIDO 1 expression was observed. In panel D and E the THP-1 cells wereexposed to zebularine for 7 days and to interferon gamma for 24 hours,either after three days (panel D) or after four days (panel E). Asustained strong IDO1 expression by the combination is illustrated inboth panel D and E.

FIG. 7: Synergistic effect on IDO expression by zebularine and twoconcentrations of interferon A, 2.5 and 25 ng/ml, respectively

FIG. 8. Synergistic effect on IDO expression by zebularine, interferongamma and interferon A

FIG. 9. Synergistic effect on IDO expression by zebularine, interferongamma and TGF-b1

FIG. 10. Sustained synergistic effect on IDO expression by zebularineand a 24 h-exposure to interferon gamma

FIG. 11. Sustained synergistic effect on IDO expression by zebularine,interferon gamma and interferon A

FIG. 12. Sustained synergistic effect on IDO expression by zebularine,interferon gamma, interferon A and TGF-beta

FIG. 13. Enhanced expression of rIdol in rat bone marrow deriveddendritic cells (BMDC) after a 5-day-exposure to 50 uM zebularine invitro and an enhanced suppressive function, inhibiting the polyclonalactivation of admixed spleen lymphocytes in vitro

FIG. 14. Zebubularine, inoculated daily for 7 days intraperitoneallyinto adult Wistar rats, induces enhanced expression of rIdol in thespleen and a suppressed T cell reactivity to polyclonal stimulation invitro

FIG. 15. Suppression of immunological rejection of allotransplantedpancreatic islets beneath the kidney capsule by daily intraperitonealinoculations of zebularine for 14 days compared to untreated controls.Blood glucose follow up on control rats as an indication of rejection.Figure shows control animal results.

FIG. 16. Suppression of immunological rejection of allotransplantedpancreatic islets beneath the kidney capsule by daily intraperitonealinoculations of zebularine for 14 days compared to untreated controls.Blood glucose follow up on zebularine treated rats as an indication ofrejection. Figure shows treated animal results.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present application and invention, the followingdefinitions apply:

The term “synergistic effect” is intended to mean an increase in the IDOlevels, after the use of a combination of IDO inducers, that issignificantly higher than the sum of the IDO levels achieved with eachof the IDO inducers if used alone, said sum usually being referred to asan “additive effect”.

The term “different mechanism of action” is defined as different ways toinduce IDO, at the molecular level and/or by more complex biologicalpathways some of which may include immunological pathways. Inparticular, it is known from the literature that the different classesof IDO inducers shown in Table 1 below have wholly or partly differentmodes of actions regarding their IDO induction. These different classesare cytidine analogues, histon deacetylase inhibitors, vitamin D3analogues, interferon gamma analogues, other interferons, such asinterferon alpha, toll like receptor ligands, gonadotropine receptorsignalling hormones, prostaglandine E2 analogues, IDO stabilizers,soluble CTLA4 conjugates, TGF-beta and glycocorticoids.

The term “immunosuppressive” is defined herein as an effect whichreduces, arrests, or ameliorates immunological insult and is protective,resuscitative or revivative for affected tissue that has sufferedcytotoxic insult from immune cells or inflammation.

The term “immunosuppressive agent” is herein defined as activeingredient or composition containing an immune insult treatment dose ofactive ingredient effective in reducing, preventing, arresting, orameliorating immune insult and provides protection, resuscitation orrevival to affected tissue that has suffered immune mediated insult oris in risk thereof.

The term “indolamine dioxygenase (IDO)” is intended to mean IDO-1(indoleamine 2,3-dioxygenase, EC 1.13.11.52), or IDO-2(indoleamine-pyrrole 2,3dioxygenase-like 1, EC 1.13.11.-) that are twodifferent proteins that can catabolize tryptophan. IDO-1 can alsocatabolize serotonin and melatonin but the substrate specificity forIDO-2 is not so well studied. Catabolites from the tryptophan pathwayare Tryptophan, N-Formyl-kynurenine, Formylanthranilate, Anthranilate,L-Kynurenine, 4-(2-Aminophenyl)-2,4-dioxybutanoate, Kynurenic acid,3-Hydroxy-L-kynurenine, 3-Hydroxy-anthranilate, 3-Metoxy-anthranilate,4-(2-Amino-3-hydroxy-phenyl)-2,4-dioxobutanoate, Xanthurenate,8-Metoxy-kurenate, 2-Amino-3-carboxy-muconate semialdehyde,2-Aminomuconate semialdehyde, Quimolinic acid, Cinnavalininate,Tryptamine, N-Methyltryptamine, Indoleacetate,2-Formamino-benzoylacetate, 5-Hydroxy-L-tryptophan,5-Hydroxy-N-formylkunerine, 5-Hydroxy-kunerine, 5-Hydroxy-kunerenamin,4,6-Dihydroxy-quinoline, Serotonin, N-Acetyl-serotonin, Melatonin,6-Hydroxy-melatonin, Formyl-N-acetyl-5-metoxykynurenamine,N-Methylserotonin, Formyl-5-hydroxy-kynurenamine, 5-Metoxytryptamine,5-Hydroxyindole-acetaldehyde, 5-Hydroxyindoleacetate,5-Metoxyindoleacetate, or 5-Hydroxyindole-acetylglycine to enhance theimmunosuppressive IDO activity. Examples are Kynurenine,3-hydroxy-kynurenine, anthranilic acid, 3-hydroxy-anthranilic acid,quinolinic acid and picolinic acid.

The immune suppression mediated by IDO is mediated by starvation oftryptophan, induction of apoptosis in lymphocytes and induction ofregulatory T-cells (Treg). The apoptosis induction and Treg induction ismediated by the catabolites, why addition of such catabolites incombination with IDO induction by composition of the invention mayenhance the clinical effect. The immune suppressive action from IDO maybe explained by 1) starvation of tryptophan, 2) direct toxic effect fromseveral of the above mentioned catabolites that induce apoptosis ofimmune cells, particularly L-Kynurenine, Anthranilate,3-Hydroxy-anthranilate and 3-Hydroxy-L-kynurenine, and/or 3) that someof the catabolites stimulate the differentiation of T helper cells toimmune suppressive regulatory T-cells important for tolerance.

An analogue is a molecule that differs in chemical structure from aparent compound, for example a homolog (differing by an increment in thechemical structure, such as a difference in the length of an alkylchain), a molecular fragment, a structure that differs by one or morefunctional groups, a change in ionization. Structural analogues areoften found using quantitative structure activity relationships (QSAR),with techniques such as those disclosed in Remington (The Science andPractice of Pharmacology, 19^(th) Edition (1995), chapter 28).

The IDO gene expression is known to be induced in antigen presentingcells. Different IDO-producing subpopulations of dendritic cells ormacrophages have a varying expression of the other enzymes in thetryptophan pathway and therefore can be anticipated to yield differentsubsets of catabolites. Dendritic cells (DC) and particularly theplasmacytoid dendritic cells (pDC) are the strongest mediators ofIDO-dependent Tcell suppression (Fallarino et al., Current DrugMetabolism 8: 209-16, 2007).

IDO expression is subject to complex regulation by an array of signals,and the IDO levels may thus be induced or maintained by differentmechanisms of actions. For example, IDO may be induced by inhibition ofDNA methyl transferases or histone deacetylases which activatesotherwise silenced promoters of IDO. IDO may also be induced by NFkBactivation which results in IDO gene expression, said NFkB activationbeing induced by various factors such as interferon gammaR1/gamma R2signaling, toll-like-receptor activation, etc.

Furthermore, inhibitors of reactive oxidative species (ROS) maycontribute to the stabilization of IDO, and so can other mechanisms thatstabilize existing IDO levels or enhance the effects of existing IDO, orinhibit pathways that degrades or inactivates IDO. Another way toincrease or maintain desired IDO levels is by inhibition of pathwaysthat are downstream other IDO inducers but which do not lead to IDOinduction, said inhibition thus favouring the IDO induction. Yet anothermechanism is by activating interferon gamma, and/or other ways toactivate an autocrine induction of IDO. These and other modes of actionfor IDO induction are described in Table 1.

The invention relates to a composition comprising at least twocompounds, which induces IDO, for the treatment of an autoimmunedisorder or disease or immune rejection of transplants or genetherapeutically modified cells, wherein said inducers have differentmechanisms of action and give rise to a synergistic effect (“synergisticeffect” being defined above) on the IDO level. Preferably, suchsynergistic effects should be significantly higher than the additiveeffect (“additive effect” being defined above), for example, at leastthree times higher. However, the synergistic effect is preferably morethan three times higher, for example, 10, 20, 30, 40, 50 or 100 timeshigher, or even more such as shown in some of the in vitro examplespresented below. Said inducers are selected from the group consisting ofcytidine analogues, histon deacetylase inhibitors, vitamin D3 analogues,Interferon gamma analogues, other interferons, toll like receptorligands, gonadotropine receptor signalling hormones, prostaglandine E2analogues, IDO stabilizers, soluble CTLA4 conjugates, andglycocorticoids. Examples of different inducers are zebularine, valproicacid, human chorionic gonadotropine and interferon gamma Other examplesare those mentioned in Table 1 below.

The invented compositions may be used for the treatment of a diseaseselected from the group consisting of Achlorhydria, Acute hemorrhagicleukencephalitis, Addison's Disease, Alopecia Areata, Anemia, PerniciousAnti-Glomerular Basement Membrane Disease, Antiphospholipid Syndrome,Aplastic Anemia, Atopic Allergy, Autoimmune Atrophic Gastritis,Autoimmune Hearing Loss, Autoimmune hemolytic anemia, Autoimmunehypoparathyroidism, Autoimmune hypophysitis, AutoimmuneLymphoproliferative, Autoimmune Myocarditis, Autoimmune oophoritis,Autoimmune orchitis, AutoimmunePolyendocrinopathy-Candidiasis-Ectodermal-Dystrophy, Autoimmune SyndromeType II, Polyglandular, Behcet Syndrome, Celiac Disease, Chagas Disease,Cholangitis, Sclerosing, Chronic Inflammatory DemyelinatingPolyneuropathy, Chronic lymphocytic thyroiditis, Churg—Strauss Syndrome,Colitis, Ulcerative, Crohn's disease, Cryoglobulinemia, CushingSyndrome, Dermatitis Herpetiformis, Dermatomyositis, Diabetes Mellitus(Insulin-Dependent), Diffuse Cerebral Sclerosis of Schilder,Encephalomyelitis, Autoimmune, Experimental (EAE), Epidermolysis BullosaAcquisita, Erythematosis, Felty's Syndrome, Glomerulonephritis (IGA),Glomerulonephritis Membranous, Goodpasture Syndrome, Graves' Disease,Guillain—Bane Syndrome, Hamman-Rich syndrome, Hepatitis Autoimmune,Hepatitis Chronic Active, Idiopathic thrombocytopenia, InflammatoryBowel Diseases, Insulin resistance—type B, Lambert—Eaton MyasthenicSyndrome, Lens-induced uveitis, Lichen Sclerosus et Atrophicus, LupusErythematosus Discoid, Lupus Erythematosus Systemic, Lupus Hepatitis,Lupus Nephritis, Lymphopenia, Meniere's Disease, Mixed Connective TissueDisease, Mooren's ulcer, Mucocutaneous Lymph Node Syndrome, MultipleSclerosis, Myasthenia Gravis, Myelitis Transverse, Myocarditis,Narcolepsy, Neuritis Autoimmune Experimental, Neuromyelitis Optica,Oculovestibuloauditory syndrome, Ophthalmia Sympathetic,Opsoclonus—Myoclonus Syndrome, Pancreatitis, Pemphigoid Bullous,Pemphigus foliaceous, Pemphigus Vulgaris, Polyarteritis Nodosa,Polychondritis Relapsing, Polyendocrinopathies Autoimmune, PolymyalgiaRheumatica, Polyradiculoneuropathy, Primary biliary cirrhosis,Psoriasis, Purpura Thrombocytopenic Idiopathic, Raynauds, ReiterDisease, Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis,Scleroderma, Sjögren's Syndrome, Spondylitis Ankylosing, Stiff—PersonSyndrome, Still's Disease Adult Onset, Takayasu's Arteritis, TemporalArteritis, Thyrotoxicosis, Type B Insulin Resistance,Uveomeningoencephalitic Syndrome, Wegener's Granulomatosis, Vitiligo.Specific examples of disearese includes Rheumatoid arthritis, Diabetesmellitus type I, Psoriasis, Sjögren's syndrome, Multiple Sclerosis,Crohn's disease, arteriosclerosis, Parkinson's disease, ALS (Amyotrophiclateral sclerosis) and dementiahe composition or in transplantations toinhibit immune rejection of organs, tissues, normal or genetherapeutically modified cells.

In particular, the invented compositions may be used for the treatmentof Rheumatoid arthritis, Diabetes mellitus type I, Psoriasis, Sjögren'ssyndrome, Multiple Sclerosis, Crohn's disease, arteriosclerosis,Parkinson's disease, ALS (Amyotrophic lateral sclerosis) or dementia.

The invented composition may further comprise a pharmaceuticallyacceptable buffer, excipient, solvent or carrier.

“Pharmaceutically acceptable” means a non-toxic material that does notdecrease the effectiveness of the biological activity of the activeingredients. Such pharmaceutically acceptable buffers, carriers orexcipients are well-known in the art (see Remington's PharmaceuticalSciences, 18th edition, A. R Gennaro, Ed., Mack Publishing Company(1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe,Ed., Pharmaceutical Press (2000).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH. Examples ofbuffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes,HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate,borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate,CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO andTES.

The term “solvent” is intended to mean an aqueous or non-aqueous liquidwith the purpose of presenting, diluting and/or dissolving thecomposition. The solvent may be one or more of saline, water,polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The excipient may be one or more of carbohydrates, polymers, lipids andminerals. Examples of carbohydrates include lactose, sucrose, mannitol,and cyclodextrines, which are added to the composition, e.g. forfacilitating lyophilisation. Examples of polymers are starch, celluloseethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose,hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates,carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid,polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone, all of different molecular weight, which are addedto the composition, e.g., for viscosity control, for achievingbioadhesion, for dilution, or for protecting the lipid from chemical andproteolytic degradation. Examples of lipids are fatty acids,phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipidsand glycolipids, all of different acyl chain length and saturation, egglecithin, soy lecithin, hydrogenated egg and soy lecithin, which areadded to the composition for reasons similar to those for polymers.Examples of minerals are talc, magnesium oxide, zinc oxide and titaniumoxide, which are added to the composition to obtain benefits such asreduction of liquid accumulation or advantageous pigment properties.

The composition may be administrated to a mammal in need thereof in asuitable amount to achieve an effect corresponding to suchconcentrations that induce a strong IDO activity in vitro of said atleast two inducers.

By having a direct effect on the immune system, treatment with asuitable dose it is also possible to use the composition forpretreatment of transplants (organs, tissues or cells) by inducing IDOexpression in the endothelial cells and as a consequence making themless immunogenic to the host and reducing the risk of rejection of thegrafted cells. By subsequent treatment of the graft recipients with thecomposition, at a dose providing immune suppression in vivo, a permanentsurvival of the transplants can be achieved without furtherimmunosuppressive therapy or with minimal such therapy.

To treat a patient, the invented composition may be administrated atdose levels that will achieve concentrations in vivo, at the sites orlocations of action, that become between 5 μM to 10 mM, or other loweror higher levels that are effective, depending on which disease ordisorder to be treated and on which IDO-inducer is referred to. ForIDO-inducers for which concentrations are normally not expressed asmolar concentrations (M), other numerical values apply, for example0.001 IU/mL to 100 MIU/mL, or more narrow ranges such as 1-1000 IU/mL,or other suitable levels. Similarly, other numerical values apply whenthe resulting levels are expressed as ng/mL, mg/mL, mg/kg body weight,etc. Initially, a higher dose may be used such as followed by a lowermaintaining dose. For the ex vivo treatment (adoptive cell transfer),similar concentrations as those outlined above should be achieved albeitthese are in vitro/ex vivo concentrations and not in vivoconcentrations. The doses aimed for, for each IDO-inducers, will also bedependent upon which other synergistically acting IDO inducer(s) that isused. Furthermore, as the number of IDO-inducers in the invented andsynergistically acting composition can be two, three, four or more, itis obvious that a very large number of feasible dose levels can bedefined.

The invented composition may be administrated by any suitable routeincluding oral, sublingual, buccal, nasal, inhalation, parenteral(including intraperitoneal, intraorgan, subcutaneous, intradermal,intramuscular, intra-articular, venous (central, hepatic or peripheral),lymphatic, cardiac, arterial, including selective or superselectivecerebral arterial approach, retrograde perfusion through cerebral venoussystem, via catheter into the brain parenchyma or ventricles), directexposure or under pressure onto or through the brain or spinal tissue,or any of the cerebrospinal fluid ventricles, injections into thesubarachnoid, brain cisternal, subdural or epidural spaces, via braincisterns or lumbar puncture, intra and peri-ocular instillationincluding application by injection around the eye, within the eyeball,its structures and layers, the ear, including the Eustachian tube,mastoid air cells, external and internal auditory canals, tympanicmembrane, middle ear, inner ear including the cochlear spiral ganglionand labyrinthine organs, as well as via enteral, bowel, rectal, vaginal,urethral or bladder cisternal. Also, for in utero and perinatalindications, then injections into the maternal vasculature, or throughor into maternal organs including the uterus, cervix and vagina, andinto embryo, foetus, neonate and allied tissues and spaces such as theamniotic sac, the umbilical cord, the umbilical artery or veins and theplacenta, may be used. The preferred route may vary depending on thecondition of the patient and the composition used in each case.

The effect of the invented composition may be combined with animmunosuppressive agent to reduce the frequency of effector immune cellsduring or before the induction of tolerance.

This invention includes the possibility of using the timing and sequenceof delivery of the invented composition to induce tolerance in anoptimal way. It also includes the possibility of using the timing andsequence of delivery of the individual IDO inducers, that comprises theinvented composition, to induce tolerance in an optimal way. Forexample, when the invented composition comprise two IDO inducers (“A”and “B”), A may be administered first, by a certain administration routeand dosage regime (dose, concentrations, frequency, etc.). Thereafter, Bwill be administered at another route, dose and dosage regime. Finally,one of A or B can be stopped before the other. A large number of suchdifferent and synergistic treatment regimes can be envisioned for thevarious combinations invented, each one taking into consideration eachIDO inducer's IDO inducing efficacy, gene expression kinetics,pharmacokinetics, etc.

The composition may comprise additional active ingredients such asmethotrexate, rapamycin, cyclophosphamide, antimetabolites includingazathioprine, inhibitors of nucleotide synthesis (includingmycophenolate mofetil, mizoribine, leflunomide, FK778), FTY720,lymphocyte depleting antibodies (including polyclonal antibodies tolymphocytes, thymocytes, T-cells, muromonab-CD3, rituximab, alemtuzumab,CAMPATH-1), non-depleting antibodies (daclizumab etc., LFA3-Ig fusionprotein), anti-TNF antibodies (including infliximab, adalimumab),natalizumab (anti-VLA-4), the anti-CD154 antibodies BG9588 and IDEC131), soluble cytokine receptors (including lenercept and etanercept(soluble TNF p55 and TNF p75 receptors), and anakinra (soluble IL-IRA).The immunosuppressive drugs mentioned above can be used in combinationwith the composition of the invention to reduce the number of immuneeffector cells.

The composition may be distributed and made available in convenient unitdosage forms such as capsules and ampoules and may be manufactured anddistributed by any of the methods known to the pharmaceutical arts. Inaddition to the active ingredient, the composition can also containother usual agents of the art relating to the type of compositionproduced. This may, by example, take the configuration of suspensions,solutions and emulsions of the active ingredient in lipid, non-aqueousor aqueous diluents, solvents, dissolving agents, emulsifiers, syrups,granulates or powders, or mixtures of these. The composition can alsocontain colouring agents, preservatives, perfumes, flavouring additionsand sweetening agents. In addition to the active ingredient, thecomposition can also contain other pharmaceutically active medications.The manufacture and distribution of the composition is carried out bytechniques known to the art, such as, evenly and intimately bringingtogether the active ingredient with liquids or fine solids or both, andthen if needed, forming the composition into a dose unit form. Thediscrete dose, portion and carrier vehicle constituting the compositionwill generally be adapted by virtue of shape or packaging for medicaladministration and distributed for this purpose.

Tablets can be manufactured and distributed by compression or mould,from active ingredient possibly with one or more additionalpharmaceutically active compounds. Compressed tablets can bemanufactured and distributed through compression in a machine typical tothe art a known quantity of the active ingredient in a dispersibleconfiguration such as powder or granules, possibly mixed with otheragents including binders, lubricants, inert diluents, preservatives, anddispersing agents. Moulded tablets can be manufactured and distributedby moulding in a machine typical to the art a mix of known quantity ofactive ingredient addition pharmaceutically active compounds and otheradditives moistened with a liquid diluent. The tablets can possibly becoated, enveloped or covered, with substances including protectivematrices, which can contain opacifiers or sweeteners and can beformulated to allow slow or controlled release, or also release within acertain part of the digestive system of the contained activeingredients. Capsules can be manufactured and distributed by placementof a known quantity of active ingredient, additional pharmaceuticallyactive compounds and additives within a two part or sealed capsule ofgelatine or other aqueous dissolvable substance. The active ingredientcan also be manufactured and distributed as a composition inmicroencapsulated, microsomal, micellar and microemulsion forms.

The compositions containing the active ingredients acceptable for oraltopical administration can be manufactured and distributed as lozengescontaining the active ingredients, other pharmaceutically activecompounds, and additives in a flavoured basis, such as acacia andtragacanth; as pastilles containing the active ingredient with otherpharmaceutically active compounds, and additives in an inert base suchas gelatine and sucrose: as mouthwashes or rinses containing the activeingredient with other pharmaceutically active compounds, and additivesin an acceptable liquid.

The composition containing the active ingredient acceptable for skintopical administration can be manufactured and distributed as ointments,oils, creams, lotions, gels, pastes and as transdermal patchescontaining the active ingredient, other pharmaceutically activecompounds, additives and an acceptable carrier medium.

The compositions containing the active ingredient acceptable for nasaladministration can be manufactured and distributed with otherpharmaceutically active compounds and additives as a powder forinhalation, or as an oily, aqueous or non-aqueous liquid for nasal sprayor drops.

The compositions containing the active ingredient acceptable for rectaladministration can be manufactured and distributed as suppositories,creams, foams, douches or enemas with other pharmaceutically activecompounds, suitable bases of the usual water-soluble diluents, fats, andadditives known to practitioners of the art.

The composition containing the active ingredient acceptable for vaginaladministration can be manufactured and distributed as pessaries,suppositories, creams, gels, foams, douches or sprays with otherpharmaceutically active compounds, suitable bases and additives known topractitioners of the art.

The composition containing the active ingredient acceptable forparenteral administration can be manufactured and distributed fromaqueous and non-aqueous sterile injection solutions, otherpharmaceutically active compounds, additives including anti-oxidants,bacteriostats and solutes and sugars such as mannitol to make thecomposition isotonic, hypotonic or hypertonic with the blood of therecipient; and also aqueous and non-aqueous sterile suspensions whichcan include suspenders and thickeners. The composition can bemanufactured and distributed in unit-dose or multi-dose containers, suchas sealed glass or plastic ampoules, vials, bottles and bags as aliquid, and in a dry state requiring only the addition of sterileliquid, for example water, saline or dextrose solutions, immediatelyprior to use. Extemporaneous solutions and suspensions for injection canbe prepared from powders and tablets of the kind above described.

The composition containing the active ingredient acceptable foradministration into the brain and related structures, spinal cord andrelated structures, ventricular system and cerebrospinal fluid spacescan be manufactured and distributed as aqueous and non-aqueous sterileinjection solutions, containing other pharmaceutically active compounds,additives including anti-oxidants, bacteriostats and solutes and sugarssuch as mannitol to make the composition isotonic, hypotonic orhypertonic with the cerebrospinal fluid; and also aqueous andnon-aqueous sterile suspensions which can include suspenders andthickeners. The composition can be manufactured and distributed inunit-dose or multi-dose containers, such as sealed glass or plasticampoules, vials, bottles and bags as a liquid, and in a dry staterequiring only the addition of sterile liquid, for example water, salineor dextrose solutions, immediately prior to use. Extemporaneoussolutions and suspensions for injection can be prepared from powders andtablets of the kind above described.

The desired unit dose of compositions, are those containing a daily doseor immune insult treatment dose or an appropriate fraction thereof, ofthe administered active ingredient. Unit dose forms of the invention mayalso include more complex systems such as double barrelled syringes,syringes with sequential compartments one of which may contain theactive ingredient, and the other any necessary diluents or vehicles. Theagents in the syringes would be released sequentially or as a mixture orcombination of the two after the triggering of the syringe plunger. Suchsystems are known in the art.

The composition may be used for the treatment of a disease or disordersuch as those mentioned above.

TABLE 1 Eleven classes of IDO-inducing substances Examples ofMechanism-of- substances in Other substances in the Action for the Classthe class class IDO induction Cytidine Zebularine, deoxy- Otherzebularine derivatives DNA methyl analogues azacytidine, and cytidineanalogues 5- transferase azacytidine methylcytidine, 2′- inhibition,deoxyzebularine, 5-fluoro- activating the zebularine, 5-fluoro-2′-otherwise silenced dexyzebularine, 5-chloro- promoters of IDO-zebularine, 5-chloro-2′- 1, and possibly of dexyzebularine, 5-bromo-FoxP3 and zebularine, 5-bromo-2′- interferon gamma dexyzebularine,5-iodo- zebularine, 5-iodo-2′- dexyzebularine, 5- methylpyrimidin-2-one,5- Me-2′-deoxyzebularine, or mono, di- or triphosphates thereof HistoneValproic acid, Other hydroxamic acids, Histon deacetylase trichostatinA, cyclic tetrapeptides (trapoxin deacetylase inhibitors vorinostat(SAHA) B) and depsipeptide, inhibition, benzamides, electrophilicactivating the ketones, phenylbutyrate, otherwise silenced belinostat(PXD 101), promoters of IDO- LAQ824, panobinostat 1 and possibly of(LBH589), CI994, FoxP3 and mocetinostat (MGCD0103) interferon gammaVitamin D3 Calcitriol Dihydrotakysterol, 1,25(OH)₂D₃ analogues(1,25(OH)₂D₃) alphacalcidol, calcitriol, inhibits Th1 (IL2, paricalcitolIFNg) and Th17 and inhibits IL12 production, all of which takes place atleast partially via IDO induction Interferon Interferon gamma Interferongamma analogues, Interferon gamma IFN-g inducers (IL12, IL18,gammaR1/gamma analogues* 4-1BB mAb) R2 signaling induced NFkB activationwhich results in IDO gene expression Other Interferon A, Interferon W1,interferon K, Interferon receptor interferons interferon B1, inducedNFkB interferon-tau activation which results in IDO gene expression Tolllike CpGcontaining DNA Other unmethylated CpG TLR-activated receptor(TLR) oligonucleotides motifs, double stranded RNA, NFkB activationligands (e.g., ODN 1826 & single stranded RNA, double- resulting in IDO2006), lipopoly- stranded unmethylated CpG- gene expression saccharidesrich DNA. Gonadotropine Recombinant human Luteinizing hormone (LH)Gonadotropine- recptor chorionic receptor signaling, signalinggonadotropine resulting in IDO hormones (rhCG), prolactin geneexpression Prostaglandins Prostaglandin E2 Other prostaglandins E2PGE2-receptor E2 analogues (PGE2) analogues, e.g., (R)-15- signaling,methyl PGE2 methyl ester, resulting in IDO (S)-15-methyl PGE2 methylgene expression ester, or 16-dimethyl PGE2 IDO stabilizers TGF-beta,TGF-beta 1:2, 1:3 and 2:3, Inhibitors of ilnterleukin-10 GDNF, BMPsReactive Oxidative Species (ROS), thereby stabilizing IDO. As well asother IDO stabilizing or IDO effect enhancing mechanisms Soluble CTLA4CTLA4-Ig Other soluble CTLA4 Binding to conjugates (abatacept/Orencia ®)conjugates CD80/86 activating interferon gamma, with an autocrineinduction of IDO Glycocorticoids Dexamethasone Other glycocorticoidInduction of GITR analogues ligand in dendritic cells and upregulationof GITR in T cells enhance the reverse signaling in DC trough GITRLinducing IDO expression.

Following examples are intended to illustrate, but not to limit, theinvention in any manner, shape, or form, either explicitly orimplicitly.

EXAMPLES Example 1 Synergistic Effect on IDO Expression by Zebularineand Interferon Gamma Materials and Methods

THP-1 (ATCC: TIB-202) is a human monocytic cell line that originatesfrom an acute monocytic leukemia. It has the phenotype of monocytes butcan be differentiated to a more dendritic phenotype. In the currentstudy, the THP-1 cells were in vitro passaged in RPMI 1640 mediumsupplemented with 5% or 10% FCS, 10 mM Hepes, 1 mM Sodium pyruvate, and50 ug/ml gentamicin (R5 or R10 medium respectively). The cell densitywas adjusted to 200 000 cells per ml, and the cultures were incubatedfor four to seven days at 37° C. with 10% CO₂ in a humidified incubator.The substance, or combination of substances, being studied, were addedto the medium at specified time points and in the case of IFN-gammaremoved from medium as indicated. 96-168 hours after initiation oftreatment, the IDO expression was assessed by PCR or by Quantitative PCR(Roche). RNA was extracted from cells cultured in flasks or six-wellplates using Trizol reagent according to Invitrogen's protocol. ResidualDNA was removed through Rnase free DNase (Roche Applied Science)treatment. Quality and quantity of the isolated RNA was measured byspectrophotometer and gel electrophoresis.

Gene expression was demonstrated using Reverse Transcriptase-PCR(RT-PCR) using the kit (Superscript one-step RT-PCR with Platinum Taq,Invitrogen) according to the instructions. The sequences of forwardprimers and reverse primers for the human IDO-1 gene analyzed and thehouse-keeping gene HPRT respectively, were: IDO-1 forward:5′-GGCAAACTGGAAGAAAAAGG-3′, reverse: 5′-CAGACAAATATA TGCGAAGAAC; HPRTForward: 5′-CAAGCTTGCTGGTGAAAAGGA-3′, HPRTReverse:5′-ACTAAGCAGATGGCCACAGAA-3′. The PCR conditions were set asfollows: 1 denaturing cycle at 94° C. for 2 min followed by 40 cycles(for the IDO-1) or 30 cycles (for the HPRT) at 94° C. for 15 s, 53° C.for 30 s, and 72° C. for 30 s with a final extension reaction at 72° C.for 5 min.

Quantitative real-time PCR analyses (qRT-PCR) were performed usingSuper-Script III Platinum Two-Step qRT-PCR Kit with SYBR Green(Invitrogen). A total of 100-500 ng total RNA was used in a 20 ul RTreaction using a mixture of polydT and random hexamer primers. The cDNAobtained was diluted to a total volume of 80 ul and stored at −20° C.The primer sequences for the different genes were designed using GeneFisher software support (G. Giegerich, F. Meyer, C. Schleiermacher,ISMB-96). The primers used for amplification of the IDO gene were:Forward: 5′-AGTCCGTGAGTTTGTCCTTTCAA-3′, Primer sequences, Reverse:TTTCACA-CAGGCGTCATAAGCT-3′.

Hypoxanthine guanine phosphoribosyl transferase (HPRT) HPRT Forward:5′-CAAGCTTGCTGGTGAAAAGGA-3′, HPRT Reverse: 5′-ACTAAGCAGATGGCCACAGAA-3′according to the cDNA sequence, were used. The qRT-PCR was performed in20 ul reaction consisting of 2 ul diluted cDNA (12.5 ng), 0.3 uM of eachprimer, 1 ul bovine serum albumin (50 ug/ml), and 10 ul Platinum SYBRGreen qRT-PCR superMix-UDG. The amplification of IDO was carried out ina Light Cycler (Roche Molecular Biochemicals) with the following thermalprofile: Platinum SYBR Green qRT-PCR superMix-UDG incubation at 50° C.for 2 min, then denaturing at 95° C. for 5 min, followed by 45 cycles at94° C. for 2 s, 58° C. for 10 s, and 72° C. for 14 s. The amplificationof HPRT was carried out as follows, UDG incubation at 50° C. for 2 min,denaturing at 95° C. for 5 min, followed by 45 cycles at 94° C. for 2 s,55° C. for 10 s, and 72° C. for 14 s. After amplification a meltingcurve analysis was performed. The qRT-PCR experiments were always run intriplicate.

Results and Discussion

Cells of the human monocytic cell line THP-1 were non-exposed (mediumcontrol), or exposed to zebularine (Berry & Associates, Inc. USA) alone,or to interferon gamma IFNg, Sigma) alone at different concentrations(FIG. 1). The cells were also exposed to 100 uM of zebularine incombination with various concentrations of interferon gamma. The scaleis logaritmic and the relative values are given for each bar. It isstriking that e.g., zebularine alone at 100 uM gave a value of 9 and 200IU/ml of inteferon gamma gave a value of 80. The sum of these values is89, whereas when both are given together the IDO1 expression reached avalue above 37 000. This demonstrates the synergistic induction of IDO1expression in THP-1 cells by the two substances.

Example 2 Synergistic Effect on IDO Expression by Interferon Gamma andValproic Acid

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to interferon gamma alone (200 IU/ml),valproic acid (Sigma) alone (1 mM) or to the combination (FIG. 2A). Theresults of THP-1 cells exposed to the combination demonstrate asynergistic effect. Similar experiments but with the valproic acidconcentration reduced to 0.5 mM was also performed (FIG. 2B). Asynergistic effect on IDO1 expression was demonstrated also with thiscombination.

Example 3 Synergistic Effect on IDO Expression by Zebularine, InterferonGamma and Valproic Acid

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to zebularine alone (100 uM), to interferongamma alone (200 IU/ml) or valproic acid alone (1 mM) (FIG. 3). TheTHP-1 cells were exposed to the three substances pairwise and also withall three substances in combination. The scale is logaritmic and therelative values are given for each bar. The result clearly demonstratesa synergistic effect of all the three substances on the IDO1 expressionin THP-1 cells.

Example 4 Synergistic Effect on IDO Expression by Human ChorionicGonadotropine (hCG) and Zebularine, and by hCG and Interferon Gamma

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to zebularine alone (100 uM), hCG (Pregnyl,Sweden) alone (0.1 units/ml), or a combination (FIG. 4A). The results ofTHP-1 cells exposed to the combination demonstrate a synergistic effect.FIG. 4B shows results when cells were non-exposed (medium control),exposed to hCG alone (0,01 units/ml), inteferon gamma alone (200 IU/ml),or a combination. The combination demonstrates a synergistic effect onIDO1 expression in THP-1 cells.

Example 5 Kinetics of IDO1 Expression after Exposure to Interferon Gammafor 24 Hours from Start and Continuous Exposure to Zebularine

Materials and Methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were exposed tozebularine alone (100 uM), interferon gamma (200 IU/ml) alone or the twosubstances in combination (FIG. 5). The aim of the study was toinvestigate the kinetics of interferon in combination with zebularine.The THP-1 cells were divided into four groups, one exposed to interferongamma alone for 24 hours, another to zebularine alone for the entireincubation period, and a third group exposed to interferon gamma for theinitial 24 hours in combination with zebularine for the entireincubation period. The THP-1 cells were washed after 24 hours andzebularine was replaced. The four groups of THP-1 cells were harvestedfor RNA isolation after 24, 48, 72 or 96 hours. The 24 hours exposure tozebularine alone gave no IDO1 induction. After 96 hours a small increasein IDO1 induction by zebularine was detected. The IDO1 induction byinterferon gamma alone was strong after 24 hours but the effect was notsustained and dropped rapidly. In contrast, when combined withzebularine maintained for the whole period, the effect was sustained atdays 2 and 3, although at a reduced level.

Example 6 Kinetics of IDO1 Expression after Interferon Gamma Exposurefor 24 Hours after Different Pre-Exposure Times to Zebularine

Materials and methods used were the same as described in Example 1above. FIG. 6 demonstrates the kinetics of synergistic effect on IDOexpression by zebularine and interferon gamma. In panel A we demonstrateby reverse transcriptase PCR (RT-PCR) a low effect of zebularine afterfour days. Interferon gamma alone given after 3 days, 24 hours beforeharvest gives a strong band and when interferon gamma is added after 3days, 24 hours before harvest of THP-1 cells exposed to zebularine, itgives a stronger effect, demonstrating a synergistic effect also whenzebularine preceeds the interferon with 3 days. In panel B, the THP-1cells were given zebularine alone during 5 days and a weak band wasdetected. Interferon gamma alone given after 3 days and washed away 24hours later, showed a weak band when tested 24 hours after removal ofinterferon. This is in agreement with results presented in FIG. 5. Thecombination of zebularine from start and interferon gamma given for 24hours after three days, resulted in a sustained high expression level.In panel C the THP-1 cells were exposed to zebularine for 6 days andinterferon gamma was given during 24 hours after four days and the cellswere harvested after a total of 6 days. Again almost no IDO1 inductionby interferon gamma alone was detected, but with the combination astrong IDO1 expression was observed. In panel D and E the THP-1 cellswere exposed to zebularine for 7 days and to interferon gamma for 24hours, either after three days (panel D) or after four days (panel E). Asustained strong IDO1 expression by the combination is illustrated inboth panel D and E. As an RNA control we have used HPRT.

Example 7 Synergistic Effect on IDO Expression by Zebularine andInterferon A

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to 100 uM zebularine (Berry & Associates, Inc.USA) alone, or to interferon A (interferon alpha, “IFN-α”, Sigma) aloneat two different concentrations, 2.5 and 25 ng/ml (FIG. 7). The cellswere also exposed to 100 uM of zebularine in combination with IFN-α atthe same two concentrations. Zebularine was present during the entireincubation, whereas IFN-α was added after 96 h and RNA isolated after120 h. The sum of the IDO1 expressions induced by zebularine alone andIFN-α alone is 3.1 and 4.7, respectively, for the lower and higher dosesof IFN-a, but for combined treatment 6.0 and 16.1 at the two IFN-α doselevels. This demonstrates the synergistic induction of IDO1 expressionby the two substances.

Example 8 Synergistic Effect on IDO Expression by Zebularine, InterferonGamma and Interferon A

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to 100 uM zebularine (Berry & Associates, Inc.USA) alone, or to 2.5 ng/ml interferon alpha (IFN-α, Sigma) alone, or to50 iu/ml interferon gamma (IFN-g) alone, and to both IFN-g and IFN-α, orfinally to a combination of all the three substances (FIG. 8). Combinedtreatment with IFN-g and IFN-α was performed by adding IFN-g to mediumat time 0 and replacing this medium at time 72 h by medium containingIFN-α, and at time 96 h replacing this medium with medium withoutadditives. Treatment with a combination of all three substances wasperformed by including zebularine and IFN-g in medium in the interval0-72 h, then replacing it with medium containing IFN-α and zebularine,and after another 24 h this medium was replaced with medium containingzebularine alone. The treatments with singular substances were performedin the same intervals, and at the end of the interval the medium wasreplaced with medium without additives. The combination of all threesubstances induces a strong synergistic effect on IDO1 expression. Thestrong synergistic effect is observed 48 h after removal of IFN-g and 24h after removal of IFN-α, indicating a sustained synergistic effect onIDO1 expression.

Example 9 Synergistic Effect on IDO Expression by Zebularine, InterferonGamma and TGF-beta

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to 100 uM zebularine (Berry & Associates, Inc.USA) alone, or to 20 ng/ml tumor growth factor beta 1 (TGF-b1) (Sigma)alone, or to 100 iu/ml interferon gamma (IFN-g), or to a combination ofIFN-g and TGF-b1, or to a combination of zebularine and IFN-g, or to acombination of all three substances, zebularine, IFN-g, and TGF-b1. Thecombination treatment with IFN-g and TGF-b1 was performed by addingIFN-g to the medium 72 h after start of culture and replacing the medium24 h later with a medium containing TGF-b1. The combination treatmentwith zebularine and IFN-g was performed by including zebularine inmedium from start of culture, after 72 h adding IFN-g and 24 h laterreplacing the medium with medium containing zebularine. The combinedtreatment with all three substances was performed by adding zebularinefrom start of culture, adding IFN-g after 72 h, and after another 24 hreplacing the medium with medium containing zebularine and TGF-b1. Thetreatments with singular substances were performed in the sameintervals, and at the end of the interval the medium was replaced withmedium without additives. The combination of all three substancesinduces a strong synergistic effect on IDO1 expression, significantlystronger than zebularine and IFN-g together or each alone (FIG. 9). Incontrast, TGF-b1, in the absence of zebularine, reduces the effect ofIFN-g. The strong synergistic effect is observed 24 h after removal ofIFN-g, indicating a sustained synergistic effect on IDO1 expression.

Example 10 Sustained Synergistic Effect on IDO Expression by Zebularineand a 24 h-exposure to Interferon Gamma

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to 100 uM zebularine (Berry & Associates, Inc.USA) alone for the entire culture period, or to 200 iu/ml interferongamma (IFN-g, Sigma) alone for 24 h (day 4 of culture) after whichculture medium was replaced with medium without additives, or to acombination of 100 uM of zebularine for the entire culture period andIFN-g for 24 h on day 4 of culture, after which culture medium wasreplaced by medium containing zebularine alone (FIG. 10). RNA wasisolated after 10, 12 or 14 days of culture and expression of IDO1 wasanalyzed. Although the expression induced by the 24 h interferon gammaexposure alone is initially strong, it is minimal at the studied timepoints. In contrast, the synergistic effect when combined withzebularine was sustained for at least 10 days after removal ofinterferon gamma, being demonstrable still at day 14. This demonstratesthat the synergistically induced IDO1 is maintained for a long time evenwhen the interferon gamma exposure is relatively short and theinterferon gamma's own effects have since long disappeared.

Example 11 Sustained Synergistic Effect on IDO Expression by Zebularine,Interferon Gamma, and Interferon A

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to 100 uM zebularine (Berry & Associates, Inc.USA) alone for the entire culture period, or to 100 iu/ml interferongamma (IFN-g, Sigma) alone for 24 h (day 4 of culture), or to interferonA (IFN-α, Sigma) alone, after which culture medium was replaced withmedium without additives. Other cell samples were exposed to acombination of 100 uM zebularine for the entire culture period of 168 hand to IFN-g, and IFN-α for 24 h on day 4 of culture, after whichculture medium was replaced by medium containing zebularine alone (FIG.11). RNA was isolated after 168 h of culture and expression of IDO1 wasanalyzed. Although the expression induced by the 24 h IFN-g or IFN-αexposure alone is initially strong, it is minimal at the studied timepoints. In contrast, the synergistic effect when combined withzebularine was sustained for 72 days after removal of the IFN-g andIFN-α. This demonstrates that the synergistically induced strong IDO1expression is maintained for a long time even when the exposure to IFN-gand IFN-α is relatively short and the own effects of IFN-g and IFN-αhave since long disappeared.

Example 12 Sustained Synergistic IDO Expression by Combined Treatmentwith Zebularine, Interferon Gamma, Interferon A, and TGF-Beta

Materials and methods used were the same as described in Example 1above. Cells of the human monocytic cell line THP-1 were non-exposed(medium control), exposed to 100 uM zebularine (Berry & Associates, Inc.USA) alone for the entire culture period, or to 100 iu/ml interferongamma (IFN-g, Sigma) alone, or to 25 ng7 ml interferon A alone, or to 20ng/ml TGF-beta (TGG-b, Sigma) alone for 24 h (day 4 of culture), afterwhich culture medium was replaced with medium without additives. Othercell samples were exposed to a combination of 100 uM zebularine for theentire culture period of 168 h and to IFN-g, IFN-α, and TGF-b for 24 hon day 4 of culture, after which culture medium was replaced by mediumcontaining zebularine alone (FIG. 12). RNA was isolated after 168 h ofculture and expression of IDO1 was analyzed. Although the expressioninduced by the 24 h IFN-g or IFN-α exposure alone is initially strong,it is minimal at the studied time points. In contrast, the synergisticeffect of the 4 substances when combined with zebularine was sustainedfor 72 days after removal of IFN-g and IFN-α. This demonstrates that thesynergistically induced strong IDO1 expression is maintained for a longtime even when the exposure to IFN-g and IFN-α and TGF-b is relativelyshort and the own effects of IFN-g and IFN-α have since longdisappeared.

Example 13 Enhanced Expression of IDO in Rat Bone Marrow DerivedDendritic Cells (BMDC) after Exposure to Zebularine In Vitro and anEnhanced Suppressive Function, Inhibiting Immune Reactivity of AdmixedSpleen Lymphocytes

Bone marrow cells were harvested from the femurs of rats and cultured byestablished in vitro techniques in the presence of the cytokines IL-4 (5ng/ml) and GM-CSF (5 ng/ml) to support differentiation into dendriticcells (BMDC) On day 7 of the culture, medium was replaced by mediumcontaining only GM-CSF. Some of the BMDC were exposed to zebularine 50uM during days 5-10 of culture and control cells were left without thisfurther treatment. The expression of IDO1 by control and zebularinetreated cells was analyzed (FIG. 13 a). The result demonstrates thatzebularine exposure enhances the expression of IDO1 above the levelexpressed by immature control BMDC. The same two types of cells werealso tested for capacity to suppress the proliferative immune responseof spleen CD4+ and CD8+ T-cells, respectively, of the same inbred strainof Fischer 344 rats upon stimulation with the strong stimulator anti-CD3attached to the bottom of culture wells. The proliferation was analyzedin FACS by the CFSE technique and the monoclonal antibodies recognizingthe CD4 and CD8 makers. Admixture of zebularine treated BMDC in aproportion of 1:30 to cultures of spleen cells demonstrated asignificantly stronger suppressive effect on the proliferative responsethan the control BMDC tested in parallel, both for CD4+ and forCD8+T-cells (FIG. 13 b). This demonstrates that exposure of BMDC tozebularine in vitro is inducing stronger expression of IDO1, and thatthese BMDC also have a stronger suppressive effect on T-cellresponsiveness than BMDC not exposed to zebularine.

Example 14 Zebularine, Inoculated Daily for 7 Days Intraperitoneallyinto Adult Rats, Induces Enhanced Expression of IDO in the Spleen and aSuppressed Spleen T Cell Reactivity to Immune Stimulation In Vitro

One group of Wistar rats was given intraperitoneal daily inoculations ofzebularine (225 mg/kg/day) for 7 days and a parallel control groupreceived intraperitoneal daily inoculations of PBS. RNA was isolatedfrom the spleens and the expression of IDO1 was analyzed by thequantitative RT-PCR technique. The results demonstrate that systemictreatment with zebularine in vivo induces enhanced expression of IDO1 inspleen cells (FIG. 14 a). Also, spleen cells from both groups wereharvested after the last dose of zebularine and tested for their T cellproliferative reactivity to polyclonal stimulation with anti-CD3antibodies. The proliferation was analyzed in FACS by the CFSE techniqueand monoclonal anti-rat CD4 and CD8 antibodies (FIG. 14 b). Thecalculated proliferative response was approximately three times lowerwith cells from zebularine-treated rats compared to cells from controlrats treated with PBS. This demonstrates that zebularine treatmentinduces enhanced expression of IDO1 in the spleen and inhibited immuneresponsiveness of spleen T lymphocytes.

Example 15 Suppression of Immunological Rejection of AllotransplantedPancreatic Islets by Daily Intraperitoneal Inoculations of Zebularinefor 14 Days

Pancreatic islets were isolated from the pancreas of Lewis rats byestablished technique. After culture at 37° C. over night, 500-1200islets were implanted beneath the kidney capsule of adult (11-14 weeksold) Fischer 344 rats that were confirmed hyperglucemic (blood glucose≧20 mMol/L) after having received a single dose of 35-40 mg/kgintraperitoneally of Streptozotocine, which is selectively toxic to theinsulin producing beta cells present in the pancreatic islets. The bloodglucose promptly decreased to normal levels as a sign of successfultransplantation of insulin producing islets. One group of these rats wasleft without further treatment as Controls, whereas another group wastreated with intraperitoneal daily inoculations of zebularine (225mg/kg) for 14 days starting 6-8 days after transplantation at a timewhen the rats had a normal blood sugar below 11.1 mMol/L. Theallotransplanted islets were immunologically rejected within 9-14 daysin 6/8 control rats (FIG. 15). The two exceptional rats that maintaineda normal blood sugar were subjected to nephrectomy 40 and 43 days aftertransplantation to check whether their normal blood glucose was a resultof unexpected sustained survival of the grafted islets (in which casetheir blood sugar should promptly increase upon removal of the graft) orwas due to a recovery of some Streptozotocine-damaged pancreatic islets(in which case their blood sugar should stay normal despite removal ofthe graft). Since the blood glucose stayed normal for ≧7 days afternephrectomy of the two control rats, it was concluded that after theinitial damage caused by Streptozotocine in these 2 rats, somepancreatic islets had recovered to produce sufficient amounts of insulinto maintain a normal blood sugar level. Twentytwo days aftertransplantation only 1/10 of the zebularine treated rats had rejectedthe graft as indicated by a normal blood sugar in all but this singlerat at completion of the treatment 20-22 days after transplantation andin all of 7 analyzed ≧1 week after the zebularine treatment was stopped(FIG. 16). This demonstrates an immunosuppressive or tolerance inducingcapacity of the ID0′-inducing substance zebularine.

1. A composition comprising at least two compounds which inducesindolamine 2,3-dioxygenase (IDO), for the treatment of an autoimmunedisorder or disease or suffering from immune rejection of organs,tissues, normal cells or gene therapeutically modified cells, whereinsaid IDO inducers have different mechanisms of action and give rise to asynergistic effect on the IDO level.
 2. The composition according toclaim 1, wherein said inducers are selected from the group consisting ofcytidine analogues, histone deacetylase inhibitors, vitamin D3analogues, interferon gamma analogues, other interferons, toll likereceptor ligands, gonadotropine receptor signalling hormones,prostaglandine E2 analogues, IDO stabilizers, soluble CTLA4 conjugates,and glycocorticoids.
 3. The composition according to claim 2, whereinsaid inducers are selected from the group consisting of cytidineanalogues, histone deacetylase inhibitors, gonadotropine receptorsignalling hormones, interferon gamma analogues, other interferons, andIDO stabilizers.
 4. The composition according to claim 1, wherein saidinducers are selected from the group consisting of zebularine, valproicacid, human chorionic gonadotropine, interferon gamma, interferon A, andTGF-beta.
 5. The composition according to claim 1, wherein saidcomposition comprising a pharmaceutically acceptable buffer, excipient,diluent, solvent or carrier.
 6. The composition according to claim 1,wherein said composition is used for the treatment of a disease selectedfrom the group consisting of Achlorhydria, Acute hemorrhagicleukencephalitis, Addison's Disease, Alopecia Areata, Anemia, PerniciousAnti-Glomerular Basement Membrane Disease, Antiphospholipid Syndrome,Aplastic Anemia, Atopic Allergy, Autoimmune Atrophic Gastritis,Autoimmune Hearing Loss, Autoimmune hemolytic anemia, Autoimmunehypoparathyroidism, Autoimmune hypophysitis, AutoimmuneLymphoproliferative, Autoimmune Myocarditis, Autoimmune oophoritis,Autoimmune orchitis, AutoimmunePolyendocrinopathy-Candidiasis-Ectodermal-Dystrophy, Autoimmune SyndromeType II, Polyglandular, Behcet Syndrome, Celiac Disease, Chagas Disease,Cholangitis, Sclerosing, Chronic Inflammatory DemyelinatingPolyneuropathy, Chronic lymphocytic thyroiditis, Churg-Strauss Syndrome,Colitis, Ulcerative, Crohn's disease, Cryoglobulinemia, CushingSyndrome, Dermatitis Herpetiformis, Dermatomyositis, Diabetes Mellitus(Insulin-Dependent), Diffuse Cerebral Sclerosis of Schilder,Encephalomyelitis, Autoimmune, Experimental (EAE), Epidermolysis BullosaAcquisita, Erythematosis, Felty's Syndrome, Glomerulonephritis (IGA),Glomerulonephritis Membranous, Goodpasture Syndrome, Graves' Disease,Guillain-Barre Syndrome, Hamman-Rich syndrome, Hepatitis Autoimmune,Hepatitis Chronic Active, Idiopathic thrombocytopenia, InflammatoryBowel Diseases, Insulin resistance—type B, Lambert-Eaton MyasthenicSyndrome, Lens-induced uveitis, Lichen Sclerosus et Atrophicus, LupusErythematosus Discoid, Lupus Erythematosus Systemic, Lupus Hepatitis,Lupus Nephritis, Lymphopenia, Meniere's Disease, Mixed Connective TissueDisease, Mooren's ulcer, Mucocutaneous Lymph Node Syndrome, MultipleSclerosis, Myasthenia Gravis, Myelitis Transverse, Myocarditis,Narcolepsy, Neuritis Autoimmune Experimental, Neuromyelitis Optica,Oculovestibuloauditory syndrome, Ophthalmia Sympathetic,Opsoclonus-Myoclonus Syndrome, Pancreatitis, Pemphigoid Bullous,Pemphigus foliaceous, Pemphigus Vulgaris, Polyarteritis Nodosa,Polychondritis Relapsing, Polyendocrinopathies Autoimmune, PolymyalgiaRheumatica, Polyradiculoneuropathy, Primary biliary cirrhosis,Psoriasis, Purpura Thrombocytopenic Idiopathic, Raynauds, ReiterDisease, Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis,Scleroderma, Sjögren's Syndrome, Spondylitis Ankylosing, Stiff-PersonSyndrome, Still's Disease Adult Onset, Takayasu's Arteritis, TemporalArteritis, Thyrotoxicosis, Type B Insulin Resistance,Uveomeningo-encephalitic Syndrome, Wegener's Granulomatosis, andVitiligo.
 7. The composition according to claim 1, wherein saidcomposition is used for the treatment of Rheumatoid arthritis, Diabetesmellitus type I, Psoriasis, Sjögren's syndrome, Multiple Sclerosis,Crohn's disease, arteriosclerosis, Parkinson's disease, ALS (Amyotrophiclateral sclerosis) or dementia.
 8. The composition according to claim 1,wherein said composition is used in transplantations to inhibit immunerejection of organs, tissues, normal cells or gene therapeuticallymodified cells.
 9. A method of inducing IDO in a cell culture comprisingthe steps of; a) providing isolated cells in a suitable medium, b)adding the composition according to claim 1, c) incubating said isolatedcells with the composition and d) obtaining a cell culture in which IDOis induced.
 10. A method of treating a mammal having an autoimmunedisorder or disease or suffering from immune rejection of organs,tissues, normal cells or gene therapeutically modified cells, whereinthe treatment induces IDO, comprising administering to a patient atherapeutically effective amount of the composition according toclaim
 1. 11. A method of treating a mammal having an autoimmune disorderor disease or suffering from immune rejection of organs, tissues, normalcells or gene therapeutically modified cells, wherein the treatmentinduces IDO, comprising firstly a treatment ex vivo of cells derivedfrom the treated mammal or from another mammal, with a therapeuticallyeffective amount of the composition according to claim 1 and in thepresence of one or more antigens associated with a condition beingtreated, followed by the transfer of treated cells to the mammal beingtreated.